The cGAS-STING pathway senses cytosolic DNA in infected cells and cancer cells and triggers production of type I interferon and other cytokines, thus triggering immune responses. Deficiency in the pathway prevents T cell responses to several viruses and to transplanted tumor lines. Our new results show that the pathway is critical for activating spontaneous NK cell responses against cancer and virus infections. Furthermore, in probing the underlying mechanisms, our research has uncovered a new cellular mechanism whereby cGAS and STING activate immune responses. Using genetic approaches, we found that the NK response to transferred tumor cells depends on cGAS expression in tumor cells and STING expression in host cells. We interpret these data to indicate that cGAS activation is the key event that occurs in infected or transformed cells resulting in the production of the cyclic dinucleotide cGAMP. cGAMP is then transferred from the infected/transformed cells to other immune cells, such as antigen presenting cells, which are induced to initiate the immune response. Hence, we propose that cGAMP transfer between cells is a fundamental mechanism in immune activation. In considering how cGAMP is transferred between cells, we hypothesized that specific membrane transporters must play a role. Using a genome-wide CRISPRi screen, we identified two transporter molecules that specifically import cGAMP into cells, one of which plays a major role and other a minor role. Using a series of knockout mice and conditional knockout mice, and cellular manipulations and transfers, we propose to test the requirement of cGAS-STING signaling in NK and T cell responses to cancer and DNA viruses, the generality of the requirement of the transfer mechanism in viral infections and cancer models and for T cell and NK cell responses, including the roles of the newly identified transporters, and to define the specific cells that must import the cGAMP for immune responses to occur. Specifically, we will: (1) Determine the sites of action of cGAMP and STING and intercellular transfer of cGAMP in anti-viral responses, including NK and T cell responses; (2) Determine sites of action of cGAMP and STING and intercellular transfer of cGAMP in T cell responses to cancer, in transfer models and GEM cancer models; (3) Determine roles of cGAMP transporters in anti-viral and anti-tumor and immune responses. Using conditional knockout mice and inhibitor studies, we will test their function in anti-herpesvirus and anti-tumor responses, including a GEM model.